This invention relates to an improved radioactive tracer which may be used in the assay of chemicals in the body such as polypeptide hormones and drugs and to methods for its preparation and use. More particularly, this invention relates to iodinated polypeptide hormone tracers which have increased sensitivity in assays, longer shelf-life and a generally improved performance during assays although their specific activities are much lower than those of tracers used heretofore. These tracers may be used in a variety of assays, including radioimmunoassays (hereafter referred to as "RIA").
The assays in which the tracers of this invention may be employed are useful for measuring the concentration of a particular substance in blood plasma, for example. For example, the assay can be used as a diagnostic test for a deficiency or overabundance of a hormone in the human body. The assay measures the blood level of the hormone and this level can be compared to the amount considered within normal bounds for individuals of similar physical characteristics. The RIA is based on the binding of antibodies to antigens, or proteins. Many antibodies are manufactured to bind to specific antigens.
The classic method for labelling polypeptide hormones, developed by Hunter and Greenwood, was originally described in Nature, Vol. 194:495 (May, 1962). Hunter and Greenwood suggest that their good results were in part, a consequence of using a low degree of iodination combined with a high specific activity.
"Specific activity" measures the amount of radioactive iodine bound to the protein which is to be used as a tracer. It has been thought that high specific activity, i.e., a high ratio of the amount of bound iodine to the amount of protein (also referred to as "antigen" or "hormone" hereafter), to which it is bound, is desirable for an accurate tracer.
The classic method for preparing tracers suitable for use in RIA is found in "A Radio-immunoelectrophoretic Assay for Human Growth Hormone," Biochemistry J. 91:43, 1964 (Hunter and Greenwood). Hunter and Greenwood employed radioactive I.sup.131. to label human growth hormone (HGH) for use in RIA. Hunter and Greenwood prepared a tracer having very high specific activity, 250-590 microcuries per microgram (uci/ug). Hunter has indicated that tracers with lower specific activities (i.e. 20-30 uci/ug) are not useful in RIAs, being badly damaged (Hunter, "the Preparation and Assessment of Iodinated 15 Antigens," Radioimmunoassy Methods, Kirkham and Hunter, 1971, p. 16).
In order to achieve the desired specific activity, a certain amount of radioactive iodine must be reacted with antigen in the initial reaction. The molar ratio of radioactive iodine to antigen will hereinafter be referred to as the "initial molar ratio." This initial ratio may also be described in terms of the ratios of absolute weights of radioactive iodine to antigen. Hunter and Greenwood used an initial weight ratio of 0.4 mci/ug protein.
It has been suggested that a high degree of iodination may deform the antigen used as a tracer, causing it to react with the antibodies to be used in the assay in a different way from unlabelled antigen, thus negatively affecting the test results. It has, therefore, been recommended that the labelled species be monoiodinated, i.e., have only one radioactive iodine atom per molecule of antigen. (J. E. Hamlin, "Radioiodination of Polypeptide Hormones," Hormones in Human Blood. Detection and Assay, Harry N. Antonaides, Ed., Harvard U.Press, Cambridge, 1976, p. 78). Thus, a recommended specific activity would lie in the range of 50 to 200 uci/ug polypeptide if 50% of the hormone in the tracer mixture is to be labelled. Hamlin also suggests using an initial molar iodine-to-hormone ratio of 0.01 to 0.20 if adequate purification can be performed, with a high specific activity (Hamlin, p.81). This preparation would contain predominantly unlabelled, or non-iodinated hormone with a small percentage of desirable monoiodinated component and no higher order derivatives which interfere with the assay.
The methods for preparing and using these tracers described by Hunter and Greenwood are time consuming. The incubation period required for the antibody-antigen reaction is prolonged due to the nature of antibody-antigen reactions.
RIA procedures performed heretofore required relatively high specific activities and, therefore, exposure to larger amounts of radiation. Exposure to radioactive iodine is dangerous because it may be inhaled and taken up by the thyroid gland, thereby causing it damage. It would be desirable to use a tracer, therefore, with smaller amounts of radioactive material. Iodinated antigens used in RIA heretofore required up to two weeks to obtain satisfactory results. In addition, tracers prepared by known classic methods tend to deteriorate quickly (approximately two weeks), necessitating frequent preparation of new tracers for use in RIAs and incurring increased exposure to radiation and great expense.
It is, therefore, an object of this invention to provide a radioactive tracer which requires less radioactive material than those used heretofore.
It is a further object of this invention to provide a radioactive tracer which is less expensive than known tracers.
It is also an object of this invention to provide a radioactive tracer which maintains at a high level its ability to function over a long period of time, using very small amounts of antigen.
It is an object of this invention to provide a rapid method of assaying polypeptide hormones in vitro.
A novel radioactive tracer composition and method for its preparation has now been discovered which has lower levels of radioactivity, is less expensive, maintains its utility over long periods of time and affords a means of determination of polypeptide hormone levels more rapid and accurate than heretofore known.